Plasma was recovered, ten fold diluted and its fluorescence emission under 450 nm excitation was measured using a spectrofluorometer (PerkinElmer LS50B, Courtaboeuf, France).
Then the cultures were ten fold diluted twice, first in MEM and subsequently in MEM supplemented with either N-hexanoyl-DL-homoserine lactone (C6-AHL) (Sigma-Aldrich, St . Louis MO, USA) or N-octanoyl-DL-homoserine lactone (C8-AHL) (Sigma-Aldrich) to a final concentration of 1 μM/mL (hereafter called MEMs).
To determine the optimal serum sample dilution, serum samples identified by the commercial SERELISA test (Synbiotics, Lyon, France) as high positive, low positive and negative were diluted in serial two fold dilutions ranging from 1 100 to 1 3200.
About 6x10 CFU/ml inoculum of M. tuberculosis was then added to the two fold serially diluted samples.
About 6×10 CFU/ml inoculum (100 μl) of M. tuberculosis was then added to the two fold serially diluted samples (100 μl).
Seven additional channels are filled with 3 mL ESD100+IIR. 3 mL of the 1,600 pg/mL standard is two fold serially diluted with 3 mL of ESD100+IIR to produce six additional standards of 800, 400, 200, 100, 50, and 25 pg/mL, leaving the eighth channel as the zero standard.
The lysate was ten fold serially diluted with PBS and 10 μl of the two highest dilutions were spotted onto Middlebrook 7H11 (MB7H11) agar plates supplemented with OADC (Becton Dickinson, USA) and 0.5% glycerol.
1.5 mL of the 20,000 pg/mL standard is three fold serially diluted with 3 mL of BlottoB to produce six additional standards of 6,667, 2,222, 741, 247, 82, and 27 pg/mL, leaving the eighth channel as the zero standard.
Milk and serum samples from these ten lactating cows were serially two-fold diluted in seven dilution steps from 1/2 up to 1/128 in BVDV antibody negative milk or serum, respectively.
